Rather than a mere storage site for triglycerides, it is now understood that adipose tissue (fat) is a complex, multicellular endocrine organ that has profound systemic effects, altering the function of nearly all other organ systems. Despite its importance, however, there is a lack of information on the dynamic nature of adipokine secretion and nutrient uptake in adipose tissue, highlighting several unmet needs in methodology. Few techniques exist to interrogate small amounts of adipose tissue, and there is a shortage of methods to explore dynamic function of the organ. Specifically, we have a limited view of the dynamic relationship between glucose, insulin, and adipose function, highlighting an immediate need for better in vitro techniques to study pancreas-adipose tissue crosstalk. As demonstrated in our previous funding period, we propose that our microfluidic systems are ideal to meet these ongoing needs. Our research team developed microfluidic approaches for culture of endocrine tissue, namely pancreatic islets and adipose tissue from C57BL/6J mice, as well as for sampling of hormone secretion. These systems permit dynamic interrogation of the tissues in ways not possible with standard techniques. The long-term goal of this research is to develop in vitro models of the endocrine system for applications in nutrition, metabolism, and drug discovery. In the short term, our objective is to develop a mouse-on-a-chip microfluidic system that permits dynamic and quantitative measurements of both hormone secretion and nutrient uptake from primary tissue. Microfluidic devices will be developed concurrently with small-volume methodology to assay secretion or nutrient uptake from pancreatic islets and adipose tissue, and 3D printing will be used to improve our novel device interfacing and tissue culture methods. Aim 1 of the proposal seeks to develop an automated microfluidic input/output multiplexer (MUX) for generalizable dynamic control over hormones and nutrients to/from endocrine tissue. Aim 2 will result in targeted small-volume compatible assays for hormones and free fatty acid uptake in adipose tissue. Aims 3 and 4 are biological in nature, using the MUX system to determine the dynamics of hormone secretion, fatty acid uptake, and lipolysis in endocrine tissues with varied glycemic dynamics (Aim 3), and determining the role of dynamic feedback between the tissues using a co-culture MUX system (Aim 4). The rationale for this research to provide a flexibly programmable, in vitro micro-model of pancreas-adipose dynamics to test several important biological hypotheses related to gut-pancreas signaling dynamics, insulin/lipolysis/fatty acid uptake dynamics, and regulation of lipolysis at low insulin and glucos (fasting or ketogenic metabolism). The proposed work is significant as a first-of-its-kind in vitro mimic of pancreas-adipose physiology, which we expect will lead to better information on human dietary interventions. The proposal is thus innovative in its technological and its biological approaches. Preliminary evidence strongly supports the feasibility of these proposals, and the research team has a proven track-record of success.